Oncogenic K-Ras signals through epidermal growth factor receptor and wild-type H-Ras to promote radiation survival in pancreatic and colorectal carcinoma cells

Abstract

Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor alpha was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers.

Figure 1

Oncogenic K-Ras expression is necessary for FTI-mediated radiosensitization. (A) DLD-1 and SG-5 cells were pretreated for 24 hours with 2.5 µmol/l tipifarnib and clonogenic cell survival assays were performed as described in Materials and Methods. DLD-1 cells express one oncogenic and one wild-type copy of K-Ras. In SG-5 cells, the oncogenic copy has been deleted by homologous recombination. (B) Clonogenic cell survival assays were performed on MiaPaCa-2 cells that were transfected with either nonspecific siRNA (control) or siRNA directed against K-Ras 48 hours before irradiation and then treated with or without 2.5 µmol/l tipifarnib 24 hours before irradiation as described in Materials and Methods. (C) Cells were treated as in (B) (without irradiation) and whole-cell lysates were analyzed by Western blot with the indicated antibody as described in Materials and Methods. C, control; K, K-Ras siRNA. (D) Western blot analysis of whole-cell lysates 48 hours after transfection with either control (con) or K-ras-specific (K-Ras) siRNA probed with antibodies specific for the K, H or N-Ras isoforms, as indicated.

Figure 2

FTI and knockdown of wild-type H-Ras expression lead to nonadditive radiosensitization of cells that express mutant K-Ras. (A) Clonogenic cell survival assays were performed using MiaPaCa-2 and DLD-1 cells that were transfected with either nonspecific siRNA (control) or siRNA directed against H-Ras 48 hours before irradiation as described in Materials and Methods. (B) Summary of the results from clonogenic cell survival assays comparing survival after transfection of pancreatic and colorectal carcinoma cells with nonspecific siRNA to survival after transfection with H-ras specific siRNA. Radiosensitization was scored positive (indicated by “+”) if the survival ratio for control/H-ras siRNA exceeded 1.1 at both 2 Gy and at 10% clonogenic cell survival. For comparison, the ability of FTI to radiosensitize these cell lines in previous studies is similarly indicated. (C) Clonogenic cell survival assays were performed on MiaPaCa-2 cells that were transfected with either nonspecific siRNA (control) or siRNA directed against H-Ras 48 hours before irradiation and then treated with or without 2.5 µmol/l tipifarnib 24 hours before irradiation as described in Materials and Methods. (D) Cells were treated as in (C) (without irradiation) and whole cell lysates were analyzed by Western blot with the indicated antibody as described in Materials and Methods. Effects of FTI on H-Ras processing are noted as unprocessed (UP) and processed (P) H-Ras proteins. C, control siRNA; H, H-ras-specific siRNA. (E) Western blot analysis of whole-cell lysates 48 hours after transfection with either control (con) or H-ras-specific (H-Ras) siRNA probed with antibodies specific for the K, H, or N-Ras isoforms, as indicated.

Figure 3

Expression of N-Ras is not necessary for FTI-mediated radiosensitization. (A) Clonogenic cell survival assays were performed on MiaPaCa-2 cells after transfection with control or N-Ras-specific siRNA 48 hours before irradiation followed by treatment with or without 2.5 µmol/l tipifarnib 24 hours before irradiation. (B) Cells were treated as in (A) (without irradiation) and whole-cell lysates were analyzed by Western blot with the indicated antibody as described in Materials and Methods. C, control siRNA; N, N-ras-specific siRNA. Effects of FTI on N-Ras processing are noted as unprocessed (UP) and processed (P) N-Ras proteins. (C) Western blot analysis of whole-cell lysates 48 hours after transfection with either control (con) or N-ras-specific (N-Ras) siRNA probed with antibodies specific for the K, H, or N-Ras isoforms, as indicated.

Figure 4

Signaling though EGFR determines radiation survival in cells that express oncogenic K-Ras. (A) Clonogenic cell survival assays were performed on MiaPaCa-2 cells that were transfected with or without 10 µmol/l gefitinib 24 hours before irradiation as described in Materials and Methods. In parallel samples (without irradiation), EGFR was immunoprecipitated and Western blot analysis with an antiphosphotyrosine antibody was performed as described in Materials and Methods. (B) Clonogenic cell survival assays were performed on MiaPaCa-2 cells after transfection with control or EGFR specific siRNA 48 hours before irradiation followed by treatment with or without 2.5 µmol/l tipifarnib 24 hours before irradiation. (C) Cells were treated as in (B) (without irradiation) and whole-cell lysates were analyzed by Western blot with the indicated antibody as described in Materials and Methods. C, control siRNA; E, EGFR-specific siRNA. (D) Clonogenic cell survival assays were performed on MiaPaCa-2 cells after transfection with control or K-Ras specific siRNA 48 hours before irradiation followed by treatment with or without 200 pmol/l TGFα 24 hours before irradiation. (E) Clonogenic cell survival assays were performed on MiaPaCa-2 cells after transfection with control or H-Ras-specific siRNA 48 hours before irradiation followed by treatment with or without 200 pmol/l TGFα 24 hours before irradiation.